Costaining of NKT cell subsets for IFN-γ and IL-17. NKT cell subsets were isolated and stimulated on anti-CD3– and anti-CD28–coated plates as described in Fig. 1. GolgiStop^TM was added to cultures 4 h before the indicated time point, and cells were stained for intracellular IFN-γ-APC and IL-17-PE. Numbers in each quadrant represent the percentage of cells in that quadrant, and results are representative of two independent experiments.

IL-17 is produced primarily by a rare population of NK1.1⁻CD4⁻ NKT cells. (A) Purified NKT cell populations from thymus and liver were stimulated as described in Fig. 1, and ICS was performed after 20 and 72 h. The first group of dot plots shows freshly isolated NKT cells for α-GC/CD1d tetramer and CD4 expression. The second and third groups represent cultured cells at 20 and 72 h, respectively, and depict CD4 versus IL-17. The numbers in each quadrant represent the percentage of total cells, except for stimulated NK1.1⁻ NKT cells, for which the numbers represent the percentage of either CD4⁺ or CD4⁻ NKT cells producing IL-17. Dot plots are representative of two experiments. (B) NKT cells from thymus and liver were purified into 4 subsets on the basis of NK1.1 and CD4 expression and cultured as described in Fig. 1. Supernatants were harvested at 24 and 72 h to assay for secreted IL-17 by ELISA. Thymus bars represent 8 separate cultures derived over four independent experiments, and liver bars represent 3–5 separate cultures derived over two independent experiments. The 24-h thymus graph contains a split y-axis. (C) NK1.1⁻CD4⁻ and NK1.1⁻CD4⁺ thymus NKT cells were assayed for intracellular IL-17 expression after 6 h CD3/CD28 stimulation in 1 experiment. Shown is IL-4 versus IL-17 expression in stimulated and nonstimulated samples. Numbers represent the percentage of cells in that quadrant.

Revised schematic of NKT cell development showing CD4⁻ NKT cells branching at the NK1.1⁻ stage.

Distinct cytokine production by NKT cell subsets. NKT cells from spleens, livers, and thymuses of B6 mice were identified as α-GC/CD1d tetramer⁺ αβTCR⁺ cells and divided further on the basis of NK1.1 and CD4 expression as shown in the upper right dot plot (shown is a representative thymus sample). Purified NKT subsets were cultured in wells coated overnight with 10 μg/ml anti-CD3 and 10 μg/ml anti-CD28. An aliquot of supernatant was harvested at 24 and 72 h to assay for secreted cytokines. IL-17 and IL-21 were assayed by ELISA. All other cytokines were assayed by cytometric bead array. The absence of bars indicates undetectable amounts of cytokine, unless “not done” is stated. For thymus and liver NKT subsets, the results are derived from 5 or 6 separate cultures collected over three independent experiments. For spleen NKT cell subsets, results are derived from 3 or 4 separate cultures collected over two independent experiments. Bars depict mean ± standard error. All cytokine values are expressed in ng/ml. Total cells = absolute cell number.

Constitutive expression of RORγt and IL-23R by NK1.1⁻ NKT cells. NKT cells were isolated from thymuses of B6 mice, separated into CD4⁺NK1.1⁺, CD4⁻NK1.1⁺, and NK1.1⁻ fractions, and stimulated for 20 or 72 h on anti-CD3/CD28–coated plates as in Fig. 1. Naïve T cells (CD4⁺/CD62L⁺/CD44^lo/CD25⁻) also were purified from spleen, and Th-17 cells were generated from naïve spleen after 3-d culture on anti-CD3–coated plates in the presence of TGF-β, IL-6, anti-IFN-γ, anti-IL-4, and 1 μg/ml soluble anti-CD28. RNA was isolated from all cell preparations, and expression of RORγt and IL-23R was determined relative to 18S ribosomal RNA. No stimulation and 20-h bars represent a total of 2 or 3 separate samples collected over two independent experiments, and 72-h bars represent 4–6 separate samples collected over three independent experiments. Bars depict mean ± standard error.

CD4⁻NK1.1⁻ NKT cells are precursors for CD4⁻NK1.1⁺ NKT cells. (A) E15 fetal thymus lobes were cultured for 1 week in standard FTOC conditions to allow development of CD4⁺CD8⁺ thymocytes. Lobes then were added to hanging drop cultures, combined with defined NKT subsets overnight, and returned to standard FTOC conditions for another 7 days before harvesting for analysis by FACS. CFSE labeling or a congenic marker together with α-GC/CD1d tetramer staining was used to identify donor NKT cells, and both doublet exclusion and unloaded tetramer exclusion were used to exclude false positives. Data are representative of 10 separate cultures for CD4⁺NK1.1⁻ NKT cell repopulations and of 9 CD4⁻NK1.1⁻ NKT cell repopulations derived over three independent experiments. (B) The percentage of mature NKT cell subsets from the given starting population that is present at the end of the culture period. Bars depict mean ± standard error. (C) NKT cells defined as α-GC/CD1d tetramer⁺ αβTCR⁺ cells were electronically gated, and NK1.1⁻ and NK1.1⁺ subsets were examined separately for expression of CD44 versus CD4. Data shown are representative of 5 separate mice for each time point.